Generation of Leishmania donovani axenic amastigotes:

their growth and biological characteristics

Alain Debrabanta, Manju B. Joshib, Paulo F.P. Pimentac, Dennis M. Dwyerb,*

aDivision of Emerging and Transfusion Transmitted Diseases, CBER, FDA, Bethesda, MD, USAbCell Biology Section, Laboratory of Parasitic Diseases, Division of Intramural Research, National Institute of Allergy and Infectious Diseases,

National Institutes of Health, Bldg. 4, Room 126, Bethesda, MD 20892-0425, USAcLaboratory of Medical Entomology, Centro de Pesquisas Rene Rachou-Fundacao Oswaldo Cruz, Belo Horizonte, Minas Gerais, Brazil

Received 14 July 2003; received in revised form 13 October 2003; accepted 21 October 2003

Abstract

In this report, we describe an in vitro culture system for the generation and propagation of axenic amastigotes from the well characterised

1S-CL2D line of Leishmania donovani. Fine structure analyses of these in vitro-grown amastigotes demonstrated that they possessed

morphological features characteristic of L. donovani tissue-derived amastigotes. Further, these axenic amastigotes (LdAxAm) were shown to

synthesise and release a secretory acid phosphatase isoform similar to that produced by intracellular amastigotes. Such LdAxAm also

expressed surface membrane 30-nucleotidase enzyme activity similar to that of tissue-derived amastigotes. Moreover, LdAxAm, in contrast to

promastigotes, expressed significant levels of the amastigote-specific A2 proteins. In addition, LdAxAm, derived from long term cultures of

Ld 1S-CL2D promastigotes, had significant infectivity for both human macrophages in vitro and for hamsters in vivo. Thus, the in vitro

culture system described herein provides a useful tool for the generation of large quantities of uniform populations of axenic amastigotes of

the L. donovani 1S-CL2D line. The availability of such material should greatly facilitate studies concerning the cell and molecular biology of

this parasite developmental stage.

q 2003 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Keywords: Trypanosomatid; Leishmaniasis; Parasite; Culture system; Infectivity; Virulence

1. Introduction

Leishmania are a group of protozoan parasites which

cause a wide range of human diseases from the localised

self-healing cutaneous lesions to fatal visceral infections

(Handman, 2001). These organisms have a digenetic life

cycle which includes extracellular, flagellated promastigote

forms that reside in the gut of their sand fly vectors and

obligate intracellular amastigote forms that reside and

multiply within the phago-lysosomal system of mammalian

macrophages. Among the numerous species of this parasite,

Leishmania donovani is the primary etiologic agent of fatal

visceral human leishmaniasis. One of the best characterised

lines of this parasite is the 1S-CL2D clone of the L. donovani

1S strain (Stauber, 1966; Dwyer, 1977). In that regard,

promastigotes of this clone (Ld 1S-CL2D) have been used to

investigate a wide variety of biochemical and biological

properties of this parasite e.g. cell surface and secreted

glycoprotein enzymes (Shakarian et al., 1997, 2002;

Shakarian and Dwyer, 1998; Debrabant et al., 2000),

lipophosphoglycan biosynthesis, structure and function

(Beverley and Turco, 1995; Descoteaux et al., 2002) and

surface membrane transporters (Vasudevan et al., 2001;

Arastu-Kapur et al., 2003; Stein et al., 2003). Such studies

were facilitated by the ability to grow large quantities of

promastigote forms of this parasite in vitro. In contrast to

promastigotes, our knowledge of the L. donovani 1S-CL2D

amastigote stage has been limited due to difficulties in

obtaining large amounts of viable amastigotes free of host

tissue contamination. Further, amastigotes isolated from

infected tissues represent heterogeneous populations at any

given time during infection, which differ presumably with

regard to their age and stage of development in their cell

cycle (Joshi et al., 1993).

To address this issue, in the current report, we describe

an in vitro culture system for the generation and continuous

0020-7519/$30.00 q 2003 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

doi:10.1016/j.ijpara.2003.10.011

International Journal for Parasitology 34 (2004) 205–217

www.parasitology-online.com

* Corresponding author. Tel.: þ1-301-496-5969; fax: þ1-301-402-2201.

E-mail address: ddwyer@niaid.nih.gov (D.M. Dwyer).

propagation of large quantities of axenic amastigotes of the

Ld 1S-CL2D strain of L. donovani. Such axenic amastigotes

were characterised with regard to their morphology (fine

structure), biochemical properties and infectivity both in

vitro and in vivo.

2. Materials and methods

2.1. Parasites

The Leishmania strains used in this study were as follows:

L. donovani strain 1S-CL2D from Sudan, World Health

Organization (WHO) designation: (MHOM/SD/62/1S-

CL2D) (Debrabant et al., 1995); L. donovani strain WR657

from India (MHOM/IN/80/DD8/WR657); L. donovani strain

WR684 from Ethiopia (MHOM/ET/67/L82/LV9/WR684);

Leishmania infantum from Spain (MCAN/SP/00/

FVM1001JL); Leishmania tropica strain WR664 from the

former Soviet Union (MHOM/SU/74/K27/,WR 664);

L. tropica strain WR 683 from the former Soviet Union

(MHOM/SU/58/OD/WR683); L. tropica strain WR646B

from Saudi Arabia (MHOM/SA/91/WR646B); Leishmania

major strain WR661, from the former Soviet Union

(MHOM/SU/73/5-ASKH/WR661); L. major strain WR662

from Israel (MHOM/IL/67/JerichoII/WR662); L. major

strain LV39 from the former Soviet Union (MRHO/SU/59/

P/LV39); L. major strain Friedlin from Israel (MHOM/IL/80/

Friedlin) and Leishmania mexicana strain M379 from Belez

(MNYC/BZ/62/M379).

Promastigotes of all these parasite strains were grown in

Medium-199 (with Hank’s salts, Gibco Invitrogen Corp.)

supplemented to a final concentration of 2 mM L-glutamine

(from 200 mM stock, Gibco), 100 mM adenosine (from

25 mM stock of free base in deionised water, Sigma

Chemical Co.), 23 mM folic acid (from 23 mM stock in

1 N KOH, Sigma), 100 IU and 100 mg/ml each of penicillin

G and streptomycin, respectively (from 10 000 IU and

10 000 mg/ml combined stock, Gibco), 1 £ BME vitamin

mix (from 100 £ stock, Sigma), 25 mM Hepes (N-[2-

hydroxyethyl]piperazine-N0-[2-ethanesulfonic acid],

Calbiochem), 4.2 mM NaHCO3 (Sigma), 10% (v/v) heat-

inactivated (45 min at 56 8C) fetal bovine serum (Gemini

Bio-Products, Woodland, CA) and adjusted with 1 N HCl

(dropwise, while stirring) to pH 6.8 at 26 8C. The final

medium (M199 þ /Hepes/pH 6.8) was sterilised by

filtration (0.45 mm, Nalgene) and stored at 4 8C prior to

use. Promastigote forms of these parasites were routinely

maintained in 5 ml of this medium in 25 cm2 Costar Brand,

plastic tissue culture flasks (Corning Inc.) at 26 8C and

transferred into fresh medium every 3–4 days as necessary.

2.2. Generation of axenic amastigotes

Promastigotes of the L. donovani 1S-CL2D strain were

also adapted to grow at 26 8C in a potassium (,140 mM)

buffered RPMI-1640 based medium. This medium was

formulated to contain the following salts (at a final

concentration of): KCl (15 mM); KH2PO4 (114.6 mM);

K2HPO4·3H2O (10.38 mM), MgSO4·7H2O (0.5 mM) and

NaHCO3 (24 mM). Other constituents of this medium were

added to a final concentration of: 1 £ RPMI-1640 vitamin

mix (from a 100 £ stock solution, Sigma); 1 £ RPMI-1640

amino acid mix (from a 50 £ stock solution, Sigma), 4 mM

L-glutamine (from 200 mM stock solution, Gibco), 100 mM

adenosine (Sigma, from 25 mM stock of free-base in

deionised water), 23 mM folic acid (Sigma, from 23 mM

stock in 1 N KOH), 100 IU and 100 mg/ml each of

penicillin G and streptomycin, respectively (from 10 000

IU and 10 000 mg/ml combined stock, Gibco), 1 £ phenol-

red (from a 1000 £ [0.5%] stock solution, Gibco), 22 mM

D-glucose (Sigma) and 25 mM 2-(N-morpholino)ethanesul-

fonic acid (MES, Calbiochem). To 1 l of this potassium-

based basal medium, 256 ml of heat-inactivated fetal

bovine serum (20% (v/v) final serum concentration, Gemini

Bio-Products) was added. The final medium was adjusted

with 2 N HCl (dropwise, while stirring) to pH 5.5 at 26 8C,

sterilised by filtration (0.45 mm, Nalgene) and stored at

4 8C prior to use. The L. donovani 1S-CL2D promastigotes

were grown in 5 ml of this medium (RPMI-1640/MES/pH

5.5 at 26 8C), in 25 cm2 Costar Brand, plastic tissue culture

flasks (Corning Inc.) at 26 8C and the ratio of culture fluid

volume to the total surface area (cm2) of the culture flask

(i.e. 1:5) was stringently maintained under all subsequent

culture conditions. These promastigotes required several

passages at relatively low dilutions (i.e. 1:10 to 1:25 of the

parasites) to adapt and grow in this acidic medium. Once

established (i.e. following four to six serial passages, each

reflecting six to eight cell divisions), these parasites were

assessed for their ability to grow in this medium at 37 8C. It

is important to point out that the medium had to be

readjusted to pH 5.5 at 37 8C since the pH of MES

containing solutions decreases with an elevation in

temperature (i.e. DpKa/8C of MES ¼ [2 ]0.011). Following

adaptation to 37 8C, parasites were grown and maintained

under the same conditions for an additional four to six

serial subcultures.

As a final step toward generating L. donovani axenic

amastigotes, these parasites were subsequently grown in

RPMI-1640/MES/pH 5.5 medium at 37 8C in a humidified

atmosphere containing 5–7% CO2 in air. Once adapted as

axenic amastigotes (LdAxAm), such parasites could be

propagated indefinitely under these conditions. In addition,

they were also fully competent to transform-back into, and

be grown as, promastigotes when placed in M199 þ

/Hepes/pH 6.8 medium at 26 8C as above. Further, this

LdAxAm cell line could be continuously cycled between

axenic amastigote and promastigote growth conditions.

Throughout this in vitro adaptation process, the cellular

morphology of the parasites was examined using both

phase-contrast light microscopy and Giemsa-stained

preparations.

A. Debrabant et al. / International Journal for Parasitology 34 (2004) 205–217206

Promastigotes of the various other Leishmania strains and

species (listed earlier) were also adapted to grow at 26 8C in

the potassium based RPMI-1640/MES/pH 5.5 medium, as

above. Typically, such promastigotes required several

passages at relatively low dilutions (i.e. 1:10 to 1:25 of the

parasites) to adapt and grow in this medium. Once

established under these conditions at 26 8C, various parasite

lines were assessed for their ability to grow at elevated

temperatures. In that regard, parasites were sequentially

incubated at increasing temperatures as necessary (e.g. 32,

34, 35, 37 or 39 8C) in the RPMI-1640/MES/pH 5.5 medium.

The medium had to be adjusted appropriately to pH 5.5 at

each of the temperatures tested. Once growing well at a

particular temperature (usually four to six serial sub-

passages) cultures were shifted to the next higher tempera-

ture. When a given parasite line reached its maximum

threshold temperature for growth, it was maintained under

the same conditions for an additional four to six serial

subcultures. As a final step toward generating ‘axenic

amastigotes’, such parasites were subsequently tested for

growth in RPMI-1640/MES/pH 5.5 medium at their

temperature optimum in a humidified atmosphere containing

5–7% CO2 in air. The cellular morphology of these parasites

was evaluated using phase-contrast light microscopy.

2.3. Growth kinetics

Parasite cultures used for growth kinetic studies were

initiated at 1–2 £ 106 cells/ml from stock cultures in their

exponential phase of growth. Aliquots of the resulting

cultures were taken at regular intervals during the time

course of their growth in vitro. Such samples were passed

through a 0.5 in., 26 gauge syringe needle five to six times to

disrupt any clumps of aggregated cells. The latter was

confirmed using phase-contrast light microscopy. Suitable

aliquots of these samples were diluted with ISOTON-II

(Coulter electrolyte balanced salt solution, Beckman–

Coulter Particle Characterisation) and counted in a Coulter

counter (Model Z1, Beckman–Coulter) equipped with a

100 mm aperture gated to a lower-threshold particle-size

limit of ^ 3 mm.

2.4. Electron microscopy

L. donovani 1S-CL2D parasites grown under various

growth conditions were harvested from log-phase cultures

(,1–2 £ 107 cells/ml) by centrifugation at 6000 £ g for

10 min at 4 8C. Cell pellets were resuspended in ice-cold PBS

(145 mM NaCl, 10 mM sodium phosphate), pH 7.2, and re-

centrifuged as above. The washed parasites were resus-

pended and fixed in 2.5% glutaraldehyde (EM grade,

Polysciences Inc., Warrington, PA) in 0.1 M sodium

cacodylate (Polysciences) buffer (pH 7.2) containing

0.146 M sucrose (Sigma), 5 mM CaCl2, for 1 h at room

temperature. Subsequently, parasites were rinsed in the same

buffer and post-fixed in 1% OsO4 in 0.1 M sodium cacodylate

containing 2 mM CaCl2, 0.8% potassium ferricyanide

(Polysciences), for 1 h at room temperature. Cells were

washed in three changes of 0.1 M sodium cacodylate,

0.146 M sucrose buffer (pH 7.2); dehydrated through an

ascending ethanol series, two changes of absolute acetone

and finally embedded in Epoxy resin (Polysciences).

Ultrathin sections were cut with an LKB Ultamicrotome

III, collected on copper grids, stained with uranyl acetate

(Polysciences) and lead citrate (Polysciences), observed and

imaged using a JEOL 100 CX transmission electron

microscope (JEOL USA) (Pimenta and de Souza, 1983).

For scanning electron microscopy (SEM), parasites

(fixed as above) were allowed to adhere on cover slips

previously coated with 0.1% aqueous poly-L-lysine (Sigma)

for 30 min at 37 8C. Subsequently, the cover slips were

washed with PBS, dehydrated with ethanol and acetone as

above. Samples were critically point dried using liquid CO2

in a Sandri-780 apparatus (Tousimis Research Corp.,

Rockville, MD) and coated with gold particles in a JFC-

110 ion-sputter device (JEOL). Samples were observed and

imaged using a JEOL-35C scanning electron microscope

(JEOL).

2.5. Metabolic labelling

Log-phase cultures (1 – 2 £ 107 cells/ml) of both

L. donovani 1S-CL2D promastigotes grown in M199 þ

/Hepes/pH 6.8 medium at 26 8C and axenic amastigotes

grown in RPMI-1640/MES/pH 5.5 medium at 37 8C and

5–7% CO2 were harvested by centrifugation at 2100 £ g for

15 min at room temperature. Cell pellets were resuspended

and washed three times by centrifugation in RPMI-1640

minus methionine (Gibco) buffered with either 25 mM

Hepes, pH 6.8 (for promastigotes) or MES, pH 5.5 (for

axenic amastigotes). Washed cells were resuspended to

2 £ 108 cells/ml in RPMI minus methionine, buffered with

either Hepes (pH 6.8 at 26 8C) or MES (pH 5.5 at 37 8C) for

promastigotes and amastigotes, respectively. L-[35S]Meth-

ionine (.800 Ci/mmol, in vivo cell labelling grade,

Amersham Biosciences) was added to a final concentration

of 25 mCi/ml and the cultures were incubated on a platform

rocker for 1 h at 26 8C for promastigotes or 37 8C with 5%

CO2 for amastigotes. All subsequent procedures were

carried out at either 4 8C or on ice. Labelled cells were

pelleted by centrifugation at 6000 £ g for 15 min. The

resulting supernatants were removed, re-centrifuged at

48 000 £ g for 30 min, made to 25 mg/ml leupeptin

(Sigma), neutralised to about pH 7 with 1 M Hepes buffer

(pH 7.4) and subsequently used for immuno-precipitation

experiments.

2.6. Immuno-precipitation of metabolically labelled

parasite culture supernatants

Cell-free 35S-labelled culture supernatants from

L. donovani 1S-CL2D promastigotes and axenic

A. Debrabant et al. / International Journal for Parasitology 34 (2004) 205–217 207

amastigotes were pre-adsorbed with Sepharose 4B beads

prior to use in immuno-precipitations as previously

described (Bates et al., 1988). A rabbit anti-serum generated

against the purified L. donovani secretory acid phosphatase

(a-SAcP Ab, rabbit # 172 (Bates and Dwyer, 1987), and

pre-immune serum from this rabbit (NRS) were used in

these immuno-precipitation experiments. For such experi-

ments, the a-SAcP Ab or NRS were first bound to pre-

washed (three times with Hepes-PBS by centrifugation)

protein-A Sepharose 4BCL beads (Amersham Biosciences)

for 1 h at 4 8C. Subsequently, these beads were washed three

times in 0.1% (v/v) Triton X-100 (Calbiochem) in Hepes-

PBS (pH 7.2) containing 25 mg leupeptin/ml. Aliquots of

such beads were then reacted with 35S-labelled culture

parasite supernatants for 1 h at 4 8C on a platform rocker.

Aliquots of the resulting protein-A antigen–antibody

complexes were washed, solubilised and analysed by

SDS-PAGE and fluorography (Doyle and Dwyer, 1993).

Similar aliquots of such immuno-precipitates were washed

and subsequently analysed for their bound secretory acid

phosphatase enzyme activity using p-nitrophenyl phosphate

as substrate as previously described (Ellis et al., 1998).

2.7. Western blotting and in situ staining of enzyme activity

Promastigotes, axenic amastigotes, and amastigotes

isolates from infected hamster spleens were lysed in

20 mM Hepes, 0.5% (v/v) Triton X-100, 25 mg/ml leupeptin

(Sigma), 100 mg/ml Aprotinin (Sigma), pH 8.0 for 30 min

on ice. Protein concentrations in total cell lysates were

determined using the bicinchoninic acid (BCA, Pierce

Chemical Co.) method (Smith et al., 1985). Subsequently,

equivalent amounts of protein from each cell lysate was

solubilised in sample buffer and analysed by SDS-PAGE

(Laemmli, 1970). Proteins were transferred onto nitro-

cellulose and processed for Western blots analysis with an

anti-Ld30NT/NU (rabbit # 1336, Debrabant et al., 1995), an

anti-A2 (Zhang and Matlashewski, 1997) or an anti-alpha

tubulin (Sigma) antibody at appropriate dilutions as

described previously (Debrabant et al., 2000). Alternatively,

SDS-PAGE gels were processed for either in situ staining of

30-nucleotidase activity according to Zlotnick et al. (1987)

or in situ staining of nuclease activity according to Bates

(1993).

2.8. Enzyme assays

30-Nucleotidase activity was measured in cell lysates of

promastigotes, axenic amastigotes and amastigotes isolated

from infected hamster spleens, in assays using 30 adenosine

mono-phosphate (30AMP, Sigma) as substrate as previously

described (Debrabant et al., 1995). 30-Nucleotidase activity

is expressed as nmol of 30AMP hydrolyzed per min per mg

of total protein (nmol/min per mg protein).

Tartrate sensitive secretory acid phosphatase activity was

measured in the cell-free culture supernatants of both

promastigotes and axenic amastigotes using p-nitrophenyl

phosphate ( pNPP) as substrate as previously described

(Shakarian et al., 2003). Results are expressed as nmol of

p-nitrophenol released from pNPP per min per ml of culture

supernatant (nmol/min per ml).

2.9. In vitro macrophage infections

The U937 human macrophage cell line used in this study

was grown in RPMI-1640 medium (Gibco Invitrogen Corp.)

supplemented with 10% (v/v) heat-inactivated fetal bovine

serum (Gemini Bio-Products) at 37 8C with 5% CO2 in air as

previously described (Doyle and Dwyer, 1993). For

experiments, U937 macrophages were grown in eight

chamber Lab-Tek tissue culture slides (Nalgene Nunc)

and infected at a 5:1 parasite to host cell ratio with either

log-phase L. donovani 1S-CL2D promastigotes or axenic

amastigotes for 2 h at 37 8C in 5% CO2. Subsequently, these

cultures were washed extensively (five times) with pre-

warmed medium to remove non-internalised parasites. One

set of such slides was immediately fixed, stained with Diff-

Quick (Dade Behring Inc.) and processed for light

microscopy (Debrabant et al., 2002). A second set of

chamber slides containing parasite infected macrophages

was incubated for an additional 72 h at 37 8C in 5% CO2.

Subsequently, these slides were fixed, stained and processed

as above for light microscope observations. Samples were

done in triplicate chambers and a minimum of 300

macrophages were counted from each chamber. Values

obtained in these experiments are expressed as the

percentage of macrophages infected by the parasites and

also as the total number of amastigotes within 100

macrophages.

2.10. In vivo infections

Male Golden Syrian hamsters (Mesocricetus auratus,

51–60 g size (,23–25 days old), Strain LVG, Charles

River Laboratories, Wilmington, MA) were infected by

intra-cardial inoculation with ,5 £ 107 L. donovani 1S-

CL2D hamster spleen (in vivo)-derived amastigotes as

described previously (Dwyer et al., 1974; Dwyer, 1976).

Intracellular L. donovani amastigotes were isolated from

heavily infected hamster spleens ,6–8 weeks p.i. The

parasite burden in such spleens was evaluated by light

microscopy using Giemsa-stained impression smears

(Dwyer et al., 1974). Typically, counts from such

Giemsa-stained preparations showed that heavily infected

spleens had amastigote burdens of .300–400 parasites

per host cell nucleus. Individual spleens from infected

animals were aseptically removed, rinsed and homogen-

ised in ice-cold PBS using a glass Ten-Broeck tissue

homogenizer (Bellco Glass, Vineland, NJ). Homogenates

were centrifuged at 250 £ g in a swinging-bucket rotor

for 10 min at 4 8C. Supernatants were removed, re-

centrifuged as above and the resulting supernatant

A. Debrabant et al. / International Journal for Parasitology 34 (2004) 205–217208

centrifuged at 2200 £ g for 20 min at 4 8C. The resulting

pellets were resuspended and wash three times with ice-

cold PBS by centrifugation as above. The final

amastigote cell pellets were resuspended in PBS, passed

through a 26 ga. syringe needle and suitable dilutions

were counted using a Petroff–Hausser bacterial counting

chamber (PGC Scientific, Gaithersburg, MD) by phase-

contrast microscopy. The latter were also verified using a

Coulter counter as described above. Counted cell

pellets were resuspended in the appropriate buffers for

use in enzyme assays, Western blots and for infecting

animals.

The infectivity of the L. donovani 1S-CL2D in vitro

grown axenic amastigotes was compared with hamster

spleen (in vivo)-derived amastigotes. To that end, two

groups of eight hamsters each were infected

intra-cardially as above with either 5 £ 107 in

vivo-spleen-derived amastigotes or log-phase in vitro-

grown axenic amastigotes. The parasite burden in

animals which succumbed to infection was evaluated,

post-mortem, using Giemsa-stained spleen impression

smears as above.

All experimental animals were housed, fed and used in

accordance with the National Institutes of Health (NIH)

Guidelines for the Care and Use of Laboratory Animals

(http://oacu.od.nih.gov). The animal study protocol was

approved by the NIH Animal Care and Use Committee.

3. Results

3.1. Development of L. donovani 1S-CL2D axenic

amastigotes

During the course of adapting the L. donovani 1S-CL2D

promastigotes to differentiate into and grow as axenic

amastigotes the parasites exhibited/assumed a variety of

morphological forms as determined by phase-contrast

microscopy and examination of Giemsa-stained parasite

preparations (Fig. 1A). This adaptation process was initiated

with cells grown at 26 8C in medium buffered to pH 6.8.

Such cells displayed a typical promastigote morphology i.e.

an elongated ellipsoidal body with an apically disposed

flagellum. When these cells were transferred and adapted to

grow at 26 8C under acidic conditions (pH 5.5) they

assumed a somewhat ‘stumpier’ (shorter) promastigote

morphology. However, when the latter cells were trans-

ferred to 37 8C under acidic conditions (pH 5.5), they

rapidly transformed (,12 h) and grew as intermediate

forms (IF). These IF parasite cultures contained a mixed

population of approximately equal numbers of amastigote-

and micromastigote/spheromastigote-like phenotypes or

morphotypes (Fig. 1A). Such parasite cultures could be

serially propagated and maintained as IF populations at

37 8C in pH 5.5 medium. However, when such IF cultures

were shifted to an atmosphere containing 5–7% CO2, the IF

Fig. 1. Generation and growth of Leishmania donovani 1S-CL2D axenic amastigotes in vitro. (A) Diagrammatic representation of the in vitro developmental

cycle of L. donovani. Promastigotes grow and proliferate at 26 8C in either pH 6.8 or 5.5 buffered medium. Parasites grown and maintained at 37 8C in pH 5.5

medium consisted of intermediate form populations (i.e. ,equal numbers of ‘amastigote’- and ‘micromastigote/spheromastigote’-like phenotypes). Cells

grown in pH 5.5 buffered medium at 37 8C with 5–7% CO2 were phenotypically axenic amastigotes. Each of the parasite phenotypes could be propagated

indefinitely ðTnÞ or cycled at will as indicated by arrows. (B) Growth curve for the various L. donovani cell phenotypes. Quadruplicate cultures were initiated

with ,106 cells/ml of each parasite phenotype and samples taken at given times for cell counting. Values represent the means of three separate determinations

for each of four cultures per time point shown. The several cell types included: promastigote phenotypes grown at 26 8C in medium buffered at pH 6.8 [-W-] or

pH 5.5 [-X-], respectively; intermediate forms grown at 37 8C in pH 5.5 medium [-B-] and axenic amastigotes grown in pH 5.5 medium at 37 8C with 5–7%

CO2 [-A-].

A. Debrabant et al. / International Journal for Parasitology 34 (2004) 205–217 209

parasites transformed into and grew as axenic amastigotes

(Fig. 1A). Once adapted to these conditions (pH 5.5 medium

at 37 8C and 5–7% CO2), such axenic amastigotes could be

continuously maintained and propagated as this phenotype.

In addition, they could also be cycled and serially

propagated, as needed, between any of the several parasite

morphological forms by altering the cell culture conditions

(i.e. pH, temperature and CO2) appropriately (Fig. 1A).

Having adapted the L. donovani 1S-CL2D parasites to

differentiate and grow as axenic amastigotes, it was of

interest to test whether this in vitro system could also be

used to generate axenic amastigotes from various other

Leishmania strains and species (i.e. those listed above). In

that regard, such parasites (previously adapted for growth at

26 8C under acidic conditions) were serially passaged and

incubated in a stepwise fashion at increasing temperatures in

RPMI-1640/MES/pH 5.5 medium. Finally parasites grow-

ing in this medium at their threshold temperature were

placed in an atmosphere containing 5–7% CO2. Following

three to four serial passages under these conditions, the

cellular morphology of such parasites was evaluated using

phase-contrast microscopy. Results obtained from those

experiments are summarised in Table 1. While the

L. donovani 1S-CL2D parasites were capable of adapting

directly from 26 to 37 8C (under acidic conditions) both, the

LV9 and DD8 strains of L. donovani required a stepwise

temperature adaptation process. However, both of these

strains eventually transformed and grew as ‘axenic

amastigote-like’ phenotypes at 37 8C, pH 5.5 with CO2.

The 1001JL strain of L. infantum required a similar

temperature adaptation process but failed to fully transform

into amastigotes at 37 8C. It is of interest to note that this

parasite line required incubation at 39 8C, pH 5.5 with CO2

to transform and grow as an axenic amastigote-like

phenotype. Of the several L. tropica strains tested

(i.e. WR664, WR683 and WR646B), all required stepwise

temperature adaptations prior to transforming and growing

as axenic amastigote-like phenotypes at 37 8C, pH 5.5 with

CO2. In contrast, the L. mexicana M379 strain parasites did

not require such a temperature adaptation process to

transform into axenic amastigotes-like phenotypes. These

parasites, once adapted to grow as promastigotes at 26 8C in

pH 5.5 medium, were capable of transforming and growing

as axenic amastigotes phenotypes in this medium when

incubated at 32 8C either in the presence or absence of

5–7% CO2. The latter observations suggest that CO2 is not

critical for the in vitro transformation of this L. mexicana

strain. Although all four L. major strains examined were

capable of growing and being serially passaged at a

threshold temperature of 35 8C at pH 5.5 with CO2, none

was able to transform into an axenic amastigote-like

phenotype under the in vitro conditions tested in this

study (Table 1). These observations suggest that L. major

must require some additional factor(s) for their in vitro

differentiation/transformation to axenic amastigotes.

While the results of the latter experiments with various

different Leishmania spp. were of interest, the main purpose

of this study was to further characterise the properties of the

axenic amastigotes generated from the L. donovani

1S-CL2D parasite line (LdAxAm). To that end, the growth

kinetics of the several different L. donovani 1S-CL2D

phenotypes (Fig. 1A) were compared during their course of

growth in vitro (Fig. 1B). Results of these analyses

demonstrated that the various parasite phenotypes/morpho-

types all had approximately the same generation/doubling

time of ,11.2 h. Moreover, when analysed during their

exponential phase of growth by flow-cytometry as described

by Doyle et al. (1991), each of these parasite phenotypes

showed a similar distribution of cells in the various phases

of cell cycle i.e. ,60, ,15 and ,25%, in G1, S and G2-M

phases, respectively (data not shown). Further, all of these

parasite cultures entered a stationary phase of growth at a

cell density of ,3–4 £ 107 cells/ml. Cumulatively, these

observations indicate that each of these cell phenotypes

appeared to possess normal growth kinetics and typical

progression through their cell cycle.

3.2. Fine structure analyses

Both scanning and transmission electron microscopy

were used to further analyse the morphological features of

the various L. donovani 1S-2D phenotypes generated in

vitro. SEM showed that parasites cultured in pH 5.5 medium

at 26 8C retained the overall characteristic shape of

promastigotes with an apically disposed flagellum

(Fig. 2A). Transmission electron microscopy (TEM)

observations verified that these parasites had the typical

overall fine structure morphology of promastigotes with an

elongate ellipsoidal body, rounded nuclei, numerous

cytoplasmic granules, apical flagellar pocket/reservoir and

Table 1

Axenic amastigotes culture conditions of various Leishmania species

Speciesa Strain Origin AxAm growth conditionsb

L. donovani 1S2D Sudan 37 8C, 5–7% CO2

LV9 Ethiopia 37 8C, 5–7% CO2

DD8 India 37 8C, 5–7% CO2

L. infantum 1001JL Spain 39 8C, 5–7% CO2

L. tropica WR664 Soviet Union 37 8C, 5–7% CO2

WR683 Soviet Union 37 8C, 5–7% CO2

WR646B Saudi Arabia 37 8C, 5–7% CO2

L. mexicania M379 Belez 32 8C, ^ 5–7% CO2

L. major WR661 Soviet Union NAc

WR662 Israel NAc

LV39 Soviet Union NAc

Friedlin Israel NAc

a All parasites were grown in RPMI-1640/MES/pH 5.5 culture medium

described in the methods section.b Axenic amastigote (AxAm) phenotype based on observations by phase-

contrast light microscopy, and Giemsa-stained preparations.c Promastigotes did not fully transform (NA) into amastigote phenotype

but were capable of growth/serial passages at 35 8C, pH 5.5, 5–7% CO2.

A. Debrabant et al. / International Journal for Parasitology 34 (2004) 205–217210

external anterior flagellum (Fig. 2D). In contrast, SEM of

parasite cultures grown at 37 8C under acidic conditions

(pH 5.5) showed that they contained a mixed population of

approximately equal numbers of amastigote- and ‘pro-/

micromastigote/spheromastigote’-like phenotypes (Fig. 2B).

The amastigote-like forms in these cultures were ovoid in

shape and lacked an external flagellum. In comparison, the

pro-/micromastigote/spheromastigote-like forms, while

somewhat ovoid in shape, still possessed an external

flagellum. These observations were verified by TEM

analyses (Fig. 2E). Further, SEM of parasites cultures

grown at 37 8C, pH 5.5 in the presence of CO2 contained

homogeneous populations of cells which displayed typical

amastigote morphology. Such parasites were ,3–5 mm in

diameter, ovoid to round in shape and had no discernible

external flagellum (Fig. 2C). However, TEM observations

showed that these parasites did possess a short flagellum that

was restricted to their flagellar pocket/reservoir (Fig. 2F).

Such parasites were morphologically indistinguishable from

amastigotes isolated from infected hamster tissues (data not

shown).

3.3. Analysis of acid phosphatase secretion

The tartrate-sensitive, secretory acid phosphatase (SAcP)

is the major secretory protein released by L. donovani

1S-CL2D promastigotes during their growth in vitro (Bates

and Dwyer, 1987). To ascertain whether SAcP was

produced and released by in vitro-grown axenic amasti-

gotes, their culture supernatants were analysed in immuno-

precipitation and enzyme activity assays. To that end,

culture supernatants from both [35S]methionine metaboli-

cally labelled promastigotes and axenic amastigotes were

reacted with a rabbit monospecific antibody (a-SAcP)

raised against the purified native, secretory acid phosphatase

of L. donovani promastigotes (Bates and Dwyer, 1987;

Bates et al., 1987). Aliquots of such immuno-precipitates

were analysed for their content by SDS-PAGE and

Fig. 2. Scanning and transmission electron microscope images of Leishmania donovani phenotypes generated in vitro. Panels (A–C) are SEM images and

panels (D–F) are TEM images. Panels (A and D): parasites grown at 26 8C in pH 5.5 medium exhibiting the typical morphology of ‘stumpy’ promastigotes.

Panels (B and E): cells cultured at pH 5.5 and 37 8C in the absence of CO2 showing mixed populations of intermediate form parasites consisting of

approximately equal numbers of amastigote- (arrow heads) and pro-/micromastigote/spheromastigote-like phenotypes. Panels (C and F): parasites grown at

37 8C, pH 5.5 in the presence of CO2 consisting of homogeneous populations of cells ,3–5 mm in diameter, ovoid to round in shape which displayed typical

amastigote morphology. Nucleus, N; granules, g; and flagellum, F. In panels A–F, bar 1 mm.

A. Debrabant et al. / International Journal for Parasitology 34 (2004) 205–217 211

fluorography as well as for their bound acid phosphatase

activity using p-nitrophenylphosphate as substrate. Results

of SDS-PAGE showed that the a-SAcP antibody specifi-

cally immuno-precipitated both the ,110 and ,130 kDa

heterodisperse isoforms of the secretory acid phosphatase

produced by L. donovani promastigotes in vitro (Fig. 3A,

lane 1). Of equal significance, was the observation that this

antibody also specifically immuno-precipitated the

.130–250 kDa heterodisperse protein band released by

axenic amastigotes into their culture supernatants (Fig. 3A,

lane 2). Further, aliquots of such immuno-precipitates were

also assayed for their bound tartrate-sensitive acid phos-

phatase enzyme activity. Results of those assays confirmed

that the a-SAcP antibody specifically immuno-precipitated

the tartrate-sensitive secretory acid phosphatase enzyme

activity produced by each of these parasite developmental

forms (data not shown). In control reactions, the pre-

immune serum (NRS) from this rabbit failed to precipitate

any labelled protein or enzyme activity from either

promastigote or axenic amastigote culture supernatants

(data not shown). The cumulative results of these short-term

metabolic labelling experiments demonstrated that both

parasite developmental forms synthesise and release distinct

heterodisperse isoforms of tartrate-sensitive secretory acid

phosphatase enzyme activity into their culture supernatants.

In light of these observations, it was of interest to

ascertain whether these two parasite developmental forms

also produced tartrate-sensitive SAcPs over their course of

growth in vitro. To that end, SAcP activity was measured in

cell-free culture supernatants obtained from both

L. donovani promastigotes and axenic amastigotes over a

time course of 96 h. As indicated earlier, under such culture

conditions, both parasite phenotypes had virtually identical

growth kinetics (i.e. see Fig. 1B), however, the amount of

SAcP activity which they released over time was signifi-

cantly different from each other (Fig. 3B). In such assays, at

equivalent cell-culture density, promastigotes appeared to

consistently release higher levels of SAcP activity com-

pared to axenic amastigotes. While cumulative results of our

immuno-precipitation and activity assays demonstrated that

both parasite developmental forms produce SAcP through-

out their course of growth in vitro both qualitative and

quantitative differences exist in the enzyme produced by

these two parasite phenotypes.

3.4. Expression of 3 0-nucleotidase/nuclease in Ld

1S-CL2DAxAm

Previously, we showed that a unique, 30-nucleotidase/

nuclease (Ld30NT/NU) was constitutively expressed on the

cell surface of L. donovani 1S-CL2D promastigotes

(Debrabant et al., 1995). It was of interest to determine

whether this enzyme was also expressed by L. donovani

amastigotes. To that end, total cell lysates of promastigotes,

axenic amastigotes, and amastigotes isolated from infected

hamster spleens (Am) were assayed for their 30-nucleoti-

dase/nuclease activities. Results of enzymatic assays

showed that lysates of promastigotes contained ,9.5- and

,14-fold more 30-nucleatidase activity than lysates of

LdAxAm or Am, respectively (Fig. 4A). Further, these

parasite lysates were also analysed by SDS-PAGE followed

by in situ staining for 30NT/NU enzymatic activities i.e.

30-nucleotidase and nuclease activities. Results showed that

a ,43 kDa band of 30-nucleotidase activity was present in

lysates of both promastigotes and LdAxAm (Fig. 4B, lane 1

and 2, respectively). However, this band of activity was

significantly less intense in lysates of LdAxAm and was not

detected in lysates of Am (Fig. 4B, lane 3). Similar results

were obtained in SDS-PAGE gels stained in situ for

nuclease activity (Fig. 4C). In order to confirm the identity

of the 43 kDa 30NT/Nu in these parasite cell lysates, they

were subjected to SDS-PAGE and Western blotting

followed by immuno-reactivity with an anti-Ld30NT/NU

specific antibody (Debrabant et al., 1995). Results of such

assays showed that the anti-Ld30NT/NU reacted with the

43 kDa Ld-30NT/Nu in lysates of promastigotes (Fig. 4D,

lane 1), as previously described (Debrabant et al., 2000).

The latter antibody also reacted with the 43 kDa Ld30NT/Nu

in lysates of LdAxAm (Fig. 4D, lane 2), however, it showed

Fig. 3. Secretion of acid phosphatase (SAcP) by Leishmania donovani

promastigotes and axenic amastigotes. (A) SDS-PAGE fluorogram of 35S-

labelled cell-free culture supernatants from promastigotes (P, lane 1) and

axenic amastigotes (Ax, lane 2) immuno-precipitated with a rabbit anti-

secretory acid phosphatase (a-SAcP) antibody. The ,110- and ,130-kDa

heterodisperse isoforms of the promastigote SAcP are marked by short

arrows on the left and the ,130–250 kDa heterodisperse enzyme

synthesised by axenic amastigotes is marked by the arrowed bracket on

the right. Molecular mass standards in kDa are indicated at the left. (B) The

production and release of tartrate-sensitive secretory acid phosphatase by

promastigotes (Pro) and axenic amastigotes (AxAm) during their time

course (in hours) of growth in vitro. Cell-free culture supernatants were

measured for SAcP activity using pNPP as substrate and are expressed as

nmol of product ( pNP) released per min per ml of parasite culture

supernatant. Values represent the mean of three separate determinations for

each of four replicate cultures per point shown.

A. Debrabant et al. / International Journal for Parasitology 34 (2004) 205–217212

no reactivity with lysates of Am (Fig. 4D, lane 3).

Consistent with our in situ staining results above (Fig. 4B

and C), the band of immuno-reactivity between the antibody

and the Ld30NT/NU was less intense in lysates of LdAxAm

than promastigotes. Taken together, these results showed

that promastigotes express significantly more Ld30NT/Nu

than either LdAxAm or Am. The level of Ld30NT/NU

expression in in vivo derived amastigotes, however,

appeared to be below the level of detection in our in situ

gel assays and Western blots.

3.5. Expression of A2 proteins in Ld 1S-CL2DAxAm

To date, a limited number of proteins have been shown to

be differentially expressed between promastigotes and

amastigotes of Leishmania. Amongst these, the A2 proteins

have been shown to be exclusively expressed in amastigotes

of L. donovani (Charest and Matlashewski, 1994). In order

to assess whether the A2 proteins were expressed by Ld

1S-CL2DAxAm total cell lysates of these axenic amasti-

gotes and of promastigotes were analysed by SDS-PAGE

and Western blotting using a specific anti-A2 antibody

(Zhang and Matlashewski, 1997). Results showed that the

anti-A2 antibody reacted with several proteins ranging from

70 to 180 kDa in lysates of LdAxAm (Fig. 5, lane 2). This

result is consistent with the reactivity of the anti-A2

antibody in Western blots with lysates of lesion-derived

amastigotes reported previously (Zhang and Matlashewski,

1997). However, the anti-A2 antibody showed only

marginal reactivity in lysates of promastigotes (Fig. 5,

lane 1). In contrast, the anti-alpha-tubulin antibody, used as

a control in these experiments, showed similar reactivity

with the lysates of both promastigotes and LdAxAm (Fig. 5,

lanes 3 and 4, respectively), indicating that similar amounts

of alpha tubulin were present in these two cell types. Taken

together, these Western blot results demonstrate that the A2

family of proteins is up-expressed by Ld 1S-CL2D axenic

amastigotes.

3.6. Parasite survival in human macrophages in vitro

Experiments were set-up to compare the infectivity of

our in vitro grown promastigotes and axenic amastigotes. In

that regard, human U937 macrophage cultures were infected

with either log-phase L. donovani 1S-CL2D promastigotes

or LdAxAm for 2 h. Subsequent to such exposure,

Fig. 4. Expression of 30-nucleotidase/nuclease by promastigotes, axenic amastigotes and lesion-derived amastigotes of Leishmania donovani. (A). 30-

Nucleotidase enzyme activity in lysates of promastigotes (P), axenic amastigotes (Ax), and amastigotes isolated from infected hamster spleens (Am). Enzyme

specific activity is expressed in nmol/min per mg total cell protein. Values reflect the mean þ SD of enzyme activity obtained from triplicate samples in two

independent experiments. (B) SDS-PAGE gel stained in situ for 30-nucleotidase activity using 30AMP as substrate. Cell lysates (20 mg) of P, Ax, and Am are

shown in lanes 1–3, respectively. (C) SDS-PAGE gel stained in situ for nuclease activity using poly-A as substrate. Cell lysates (20 mg) of P, Ax, and Am are

shown in lanes 1–3, respectively. (D) Western blots showing the reactivity of cell lysates (15 mg) of P, Ax, and Am (lanes 1–3, respectively) with an anti-

Ld30NT/NU specific antibody. Molecular mass standards in kDa are indicated at the left of panels B–D.

Fig. 5. Expression of A2 proteins by axenic amastigotes and promastigotes

of Leishmania donovani. Western blots of cell lysates (20 mg) of

promastigotes (P) and axenic amastigotes (Ax), reacted with either anti-

A2 (A2, lanes 1 and 2) or anti-alpha-tubulin (Tubulin, lanes 3 and 4)

specific antibodies. Molecular mass standards in kDa are indicated at the

left.

A. Debrabant et al. / International Journal for Parasitology 34 (2004) 205–217 213

the percentage of infected macrophages and the number of

parasites/100 macrophages was determined by light

microscopy of stained preparations. Such observations

indicated that similar numbers of each parasite phenotype

were taken up by the U937 macrophages after 2 h of contact

(i.e. ,60–70% of macrophages were infected). In addition,

when such macrophages were scored for their parasite load

no significant differences were observed between those

exposed to promastigotes or LdAxAm (i.e. 169 and 139

parasites per 100 macrophages, respectively). After 72 h of

incubation, the parasite load within these macrophages was

determined. Results of these assays showed that macro-

phages infected with LdAxAm had a significantly higher

parasite load than those exposed to promastigotes (Fig. 6A).

Taken together, these observations indicated that axenic

amastigotes survived significantly better than promastigotes

in U937 macrophages.

3.7. Infectivity of Ld 1S-CL2DAxAm in vivo

In light of our in vitro results with U937 macrophages,

experiments were set-up to test the infectivity of axenic

amastigotes in vivo. To that end, hamsters were inoculated

intracardially with either Ld 1S-CL2D AxAm or tissue-

derived amastigotes. Such animals were monitored for their

response over time. Hamsters inoculated with tissue-derived

amastigotes uniformly developed fatal visceral infections

and all succumbed to the parasite between 7 and 10 weeks

p.i. (mean ,58 days) (Fig. 6B). In parallel experiments,

most of the hamsters inoculated with in vitro grown

LdAxAm also developed similar fatal visceral infections;

however, the onset of their symptoms was considerably

delayed. In that regard, 75% of the animals in this group

(i.e. six out of eight) succumbed to infection between 9 and

16 weeks (mean ,89 days) (Fig. 6B). At 18 weeks p.i., the

two remaining animals in this group were sacrificed and

found to have severe parasite burdens in their spleens

(.500 amastigotes/host cell nucleus) typical of fatal

visceral infections. Results of this experiment showed that

in vitro-generated axenic amastigotes of L. donovani

1S-CL2D were capable of producing fatal visceral infec-

tions in hamsters.

4. Discussion

In the current report, we have described an in vitro

culture system for the generation and continuous propa-

gation of large quantities of axenic amastigotes from the Ld

1S-CL2D cloned line of L. donovani. This in vitro culture

system was devised to mimic some of the environmental

conditions which intracellular L. donovani amastigotes

would encounter within the phago-lysosomal system of

macrophages in vivo (e.g. acidic pH, elevated CO2 and

temperature, high potassium/low sodium milieu, etc.).

When adapted to grow under such conditions, Ld 1S-

CL2D promastigotes assumed an amastigote-like phenotype

which could be continuously propagated in vitro. Results of

SEM and TEM fine structure observations demonstrated

that such in vitro-grown amastigotes (LdAxAm) possessed

the morphological features characteristic of L. donovani in

vivo/tissue-derived amastigotes. In addition, once esta-

blished as axenic amastigotes, these cells could also be

Fig. 6. Infectivity of LdAxAm in vitro and in vivo. (A) Parasite burden in human U937 macrophages. Macrophages were examined 72 h after infection with

either Leishmania donovani promastigotes (Pro) or axenic amastigotes (AxAm). The parasite burden is expressed as the number of intracellular amastigotes per

100 macrophages with error bars indicating the standard deviations (SD). Values reflect the mean þ SD of counts obtained from triplicate samples in two

independent experiments. (B) Comparison of the infectivity of L. donovani tissue-derived amastigotes versus axenic amastigotes in hamsters. Two groups of

eight hamsters each ðn ¼ 8Þ were inoculated intracardially with either in vivo-derived amastigotes (i.e. [Am, -B-] freshly isolated from infected hamster

spleen) or in vitro generated axenic amastigotes (AxAm, -A-). The survival of these animals was monitored daily over a period of 18 weeks post-infection. *On

necropsy at 18 weeks p.i. the two remaining animals in this group had extremely heavy intracellular parasite burdens in their spleens (.500 amastigotes per

host cell nucleus), a characteristic marker of fatal visceral disease in these animals.

A. Debrabant et al. / International Journal for Parasitology 34 (2004) 205–217214

cycled, at will, between the LdAxAm and promastigote

phenotypes by altering the culture conditions.

In this study, we also observed that the change in parasite

phenotype from promastigote to axenic amastigote corre-

lated with changes in protein expression. For example,

results of biochemical analyses showed that both promas-

tigotes and LdAxAm synthesised and released tartrate-

sensitive secretory acid phosphatase enzyme activity.

However, each of the parasite developmental forms

appeared to produce its own unique heterodisperse isoform

of this enzyme. The difference between these two SAcP

isoforms may be due to specific post-translational modifi-

cations (e.g. type and/or amount of glycosylation, phos-

phorylation, etc.) to this enzyme unique to each parasite

developmental form. Further, the heterodisperse isoform of

SAcP produced by LdAxAm is similar to that produced by

Ld 1S-CL2D amastigotes within infected U937 macro-

phages as reported previously (Doyle and Dwyer, 1993).

Moreover, our results showing that LdAxAm produce SAcP

are also in agreement with previous observations which

demonstrated that L. donovani amastigotes synthesise SAcP

during the course of human infections (Ellis et al., 1998).

As part of the biochemical characterisation of LdAxAm,

they were analysed for their expression of surface

membrane bound 30-nucleotidase-nuclease activity. Those

results showed that LdAxAm constitutively expressed low

levels of 30NT/NU activity compared to promastigotes.

However, the amount of enzyme activity produced by

LdAxAm was similar to that produced by amastigotes

isolated from infected hamster spleen tissue. Taken

together, these results are in good agreement with

experiments which showed that L. donovani amastigotes

synthesise 30-nucleotidase-nuclease activity during the

course of human visceral leishmaniasis infections (Dwyer,

unpublished).

To further characterise the LdAxAm, they were analysed

for their expression of A2 proteins. This family of proteins

has been shown to be developmentally up-expressed in

amastigotes of L. donovani (Charest and Matlashewski,

1994). In agreement with the latter, our results showed that

these A2 proteins were predominantly up-expressed in

LdAxAm compared to promastigotes. These observations

suggest that the culture conditions described in this report

must provide adequate signals to trigger the up-expression

of the A2 proteins in axenic amastigotes. Taken together,

the results of our biochemical assays showed that the Ld

1S-CL2D AxAm possessed characteristics similar to those

of in vivo-derived amastigotes.

With regard to infectivity, in the current report, we

showed that LdAxAm, derived from long term cultures of

Ld 1S-CL2D promastigotes, had significant infectivity in

vitro in human macrophages as well as in vivo in

hamsters. Although LdAxAm were not as virulent as

amastigotes isolated from infected spleens, they did cause

fatal visceral disease in experimentally infected hamsters. In

contrast, hamsters inoculated with the parental, long-term

(.600 serial passages) in vitro cultured Ld 1S-CL2D

promastigotes showed no evidence of infection over a

similar time course. Cumulatively, these results demon-

strated the infectivity of the in vitro generated LdAxAm.

To date, a number of in vitro culture systems have been

described for the generation of axenic amastigotes from

various different leishmanial stains and species (for refer-

ences see comprehensive review by Gupta et al., 2001). The

culture system described in the current report was primarily

developed to generate axenic amastigotes from the well

studied 1S-CL2D cloned line of L. donovani. In addition, this

in vitro culture system was also used to generate axenic

amastigote-like forms from various other Leishmania strains

and species. With the exception of L. major, all the other

parasites tested acquired an amastigote-like phenotype as

assessed by light microscopy. However, the biological and

biochemical properties of these axenic amastigote-like forms

remain to be established experimentally.

In summary, in the current report, we describe an in vitro

culture system for the generation of axenic amastigotes from

1S-CL2D line of L. donovani. Such amastigotes were

characterised with regard to some of their biological and

biochemical properties. This in vitro culture system

provides a useful tool for the generation of large quantities

of uniform populations of axenic amastigotes of this

parasite. The availability of such material should greatly

facilitate studies concerning the cell and molecular biology

of this parasite developmental form. In that regard, such

LdAxAm have already been used to elucidate, for example,

the differential/developmental expression of some

L. donovani genes and their products (Joshi et al., 1993,

1996; Debrabant et al., 1995; Pogue et al., 1995; Ghedin

et al., 1998; Duncan et al., 2001; Selvapandiyan et al., 2001;

Shakarian et al., 2002; Padilla et al., 2003). In addition,

these axenic amastigotes have also been used to examine

programmed cell death pathways in L. donovani (Lee et al.,

2002; Debrabant et al., 2003), in high throughput screening

assays to identify novel anti-leishmanial compounds (Pitzer

et al., 1998; Havens et al., 2000; Brendle et al., 2002) and

recently in developmental and genetic studies of L. donovani

phosphoglycans (Goyard et al., 2003).

In conclusion, axenic amastigotes of L. donovani

1S-CL2D constitute a valuable research tool to further

investigate the unique biological properties of this lethal

human pathogen.

Acknowledgements

We thank Dr Greg Matlashewski (Department Micro-

biology and Immunology, McGill University, Montreal,

CA) for providing the anti-A2 antibody used is this study.

Dr Joshi was supported by Postdoctoral Intramural Research

Training Award Fellowship from the NIAID, NIH.

A. Debrabant et al. / International Journal for Parasitology 34 (2004) 205–217 215

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